Peptide sample enrichment using μPAC™ Trapping columns
The online preconcentration of analytes onto low volume trapping columns is a commonly used injection strategy in nano- and microbore LC-MS/MS analysis of complex peptide mixtures. Compared to direct injection onto the analytical column, a sample trapping approach provides several advantages. By effectively desalting and preconcentrating the analytes of interest onto the trap column, analytical column lifetime and workflow throughput can be improved. The trapping configuration allows effective removal of sample matrix components such as salts and contaminants which can interfere with downstream MS analysis, hereby increasing analytical column lifetime and at the same time improving detection sensitivity and the spectral quality that is generated for a sample. It also allows loading higher volume and dilute samples at a much higher flow rate than what is feasible when working with a direct injection approach, having a positive effect on the LC-MS/MS duty cycle.
However, apart from providing sample clean-up and preconcentration, it is also of paramount importance to maintain chromatographic performance when combining an analytical column with a trapping column. A poor combination will result in reduced chromatographic performance, which will in turn affect the quality of the data that is generated. Trapping column dimensions and surface chemistry have to be selected carefully to match the analytical column that is used. Typically, trapping columns with a capacity factor slightly lower than the analytical column will result in the best chromatographic performance as analytes will experience a second preconcentration or refocusing event when eluted onto the analytical column. This eliminates the detrimental effect of pre-analytical column connections or void volumes on the separation eficiency.
Compared to conventional packed bed nano LC columns, the stationary phase support of the μPAC™ columns consists of superficially porous silicon pillars that have been modified with a hydrophobic ligand (Octadecyl or C18), and therefore they have a slightly lower capacity factor. Combining traditional ‘fully porous’ packed bed trapping columns with a μPAC™ analytical column will consequently result in decreased chromatographic performance.
To support the use of the 50 and 200 cm long μPAC™ columns, a micromachined trapping column was developed with matching stationary phase support morphology. Optimal trapping configuration and sample loading conditions have been evaluated and compared towards a direct injection strategy in terms of chromatographic performance and protein identifications that can be achieved in a typical bottom-up proteomics experiment.